| In this study,a dual-mode molecular probe cRGD-Gd-Cy5.5 targeting integrin alpha v beta 3 was constructed,and its characterization,in vivo and in vitro targeting properties were discussed,which provided a theoretical basis for the molecular imaging study of nasopharyngeal carcinoma and a reference for further target therapy.Objective: To investigate the preparation of a dual-mode molecular probe cRGD-Gd-Cy5.5 targeting integrin alpha v beta 3 by magnetic resonance imaging(MRI)/near infrared fluorescence imaging(NIRF)and the feasibility of targeting integrin alpha V beta 3 tumor cells in vitro and in vivo.Method:(1)Gadolinium trichloride liposomes were synthesized by thin film dispersion method,and RGD-Gd-Cy5.5 molecule was coupled to the surface of the liposomes to obtain a dual-mode molecular probe cRGD-Gd-Cy5.5.(2)The particle size of the probe was measured by transmission electron microscopy(TEM);the hydration particle size and zeta potential were measured by dynamic light scattering(DLS);and the fluorescence coupling effect was detected by multifunctional enzyme label.(3)The fluorescence and magnetic resonanceimaging capabilities of cRGD-Gd-Cy5.5 in EP tube were observed by fluorescence imaging system and 3.0T MR imaging system.The probe in EP tube was scanned by T1 WI sequence to obtain T1 relaxation time and calculate T1 relaxation rate.(4)CCK8 method was used to detect the cytotoxicity of the probe in vitro,and healthy nude mice model was used to detect the acute toxicity of the probe in vivo.(5)Laser confocal microscopy was used to verify the cell targeting of cRGD-Gd-Cy5.5 in vitro: A549,SUNE-1-5-8F,MCF-10 A cell lines were co-incubated with molecular probes,and the fluorescence distribution characteristics were observed after washing more than the probes and compared with the results of cellular immunofluorescence.(6)Establishment of a nude mice model of nasopharyngeal carcinoma xenografts and validation of in vivo targeting of cRGD-Gd-Cy5.5 magnetic resonance using the model: The nude mice model of nasopharyngeal carcinoma xenografts was randomly divided into target group and non-target group.RGD-Gd-Cy5.5(target group)and Gd-DTPA(non-target group)were injected via tail vein respectively.T1 weighted imaging was performed before and 0.5,2,4 and 6hours after injection,respectively.The tumors and their adjacent normal tissues were used as regions of interest(ROI)to measure signal intensity,and the signal-to-noise ratio(SNR)of magnetic resonance images was calculated,and the SNR of the two groups of images was compared.(7)The fluorescence in vivo targeting of the probe was validated by small animal fluorescence imaging system.The transplanted model of nasopharyngeal carcinoma in nude mice was randomly divided into target group and closed group.The target group was injected with cRGD-Gd-Cy5.5 probe solution(concentration 300 umol/kg [Gd])via tail vein,and the closed group was injected with excessive free cRGD polypeptide molecule through tail vein to close the binding site of integrin alphav beta 3,waiting for 2 hours before passing through tail.In vivo fluorescence scanning was performed at 30 min,1 h,2 h,4 h and 6 h after intravenous injection of the same concentration of probe solution.The difference of probe concentration at tumor sites between the two groups was observed.(8)After fluorescence imaging of the nude mice tumor model was completed,the nude mice were executed and the tumor tissue sections were taken to verify the histological targeting of cRGD-Gd-Cy5.5 to integrin alpha v beta 3 by immunofluorescence staining.Results:(1)The preparation of cRGD-Gd-Cy5.5 was successful.(2)The particle size of cRGD-Gd-Cy 5.5 was 60.08(±0.45)nm,the hydrodynamic size was 95.58(±8.31)nm,the zeta potential was 28.9(±1.32)mV,and the maximum absorption peak at 685 nm was red fluorescence.(3)The relaxation rate of cRGD-Gd-Cy 5.5 T1 was 10.515 mm-1S-1 measured by magnetic resonance.(4)In vitro cytotoxicity test,the survival rate of cRGD-Gd-Cy 5.5 was more than 70% even at Gd concentration of 400 ugM,showing a low cytotoxicity,and there was no acute toxicity in animal toxicity test by H-E staining.(5)Confocal microscopy confirmed the cell targeting ability of the probe to high expression of integrin alpha v beta 3 cells.The probe was mainly distributed on the surface of cell membrane,which was consistent with the expression site of integrin alpha v beta 3.(6)Magnetic resonance imaging of nasopharyngeal carcinoma xenografts in nude mice proved that cRGD-Gd-Cy5.5 had good targeting in vivo and obtained a higher signal-to-noise ratio of magnetic resonance than Gd-DTPA(p < 0.05),with the highest SNR of 3.22.(7)In vivo fluorescence imaging targeting group,the signal of tumor location was stable and persistent,but there was no specific fluorescence aggregation in target closure group.(8)Under laser confocalmicroscopy,the histological distribution of cRGD-Gd-Cy5.5 is regular,and it has the same histological localization as the monoclonal antibody of integrin alpha v beta 3.The histological targeting of cRGD-Gd-Cy5.5 was successfully verified by tissue immunofluorescence staining.Conclusion: Dual-mode molecular probe cRGD-Gd-Cy5.5 has good characterization,stability,biological safety,high T1 relaxation rate and strong binding ability to target integrin alpha v beta 3,which provides a broad application prospect for imaging diagnosis of nasopharyngeal carcinoma targeting integrin. |
PDF Full Text Request